Method of preparing integral multilayer analytical element

ABSTRACT

A method of preparing an integral multilayer analytical element such as for analysis of calcium comprising a water-impermeable light-transmissive support, a reagent layer containing a water-soluble indicator capable of reacting with an analyte to produce an optically detectable change, and a porous spreading layer containing a spreading action controller and/or an acid capable of decomposing the calcium compounds in a sample, superposed in this order, wherein the spreading action controller and/or the acid is dissolved in an organic solvent which does not dissolve the above water-soluble indicator, this solution is incorporated into the porous spreading layer, and this superposed material is dried. In the method of the present invention, the migration of the water-soluble indicator in the reagent layer at the time of incorporating the spreading action controller or the acid into the spreading layer is inhibited by using a particular organic solvent. Migrations of the indicator and colored material to the spreading layer are also lowered by the spreading action controller, and by these, analytical accuracy of the analytical element is improved. In the case of the analytical element for analysis of calcium, permeation of the acid into other layers than the spreading layer is inhibited, and permeation of a pH buffer in other layer(s) into the spreading layer is also inhibited by using a particular organic solvent. As a result, effective decomposition of calcium compounds in a sample and effective coloration can be achieved.

This is a divisional of Ser. No. 073,759, filed 7-15-87, now U.S. Pat.No. 4,871,679.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of preparing an integral multilayeranalytical element for analysis of an analyte in a liquid sample. Moreparticularly, this invention relates to an improvement of a method ofpreparing a dry-type integral multilayer analytical element for analysisof an analyte in a liquid sample, such as, a biological body fluid,including blood (whole blood, blood plasma, blood serum), cerebrospinalfluid, lymph, saliva and urine, and which is useful for diagnosis in theclinical field.

2. Description of the Prior Art

Various integral multilayer analytical elements have been known.However, in every analytical element, when a water-soluble substance isused as the indicator for reacting with the analyte in a sample togenerate an optical change, the indicator diffuses into the upper porousspreading layer and results in a lowering of the analytical accuracy.(This diffusion is called "migration".) In the case that the indicatoris highly soluble in water, such as in the case of the analyticalelement for analysis of calcium, the analytical accuracy is remarkablylowered by the migration. Since the change in the calcium concentrationin blood does not vary widely, this defect is fatal of this analyticalelement.

Therefore, various investigations have been made in order to eliminatethe defect. For example, a migration-inhibiting layer can be introducedinto the analytical element as disclosed in U.S. Pat. No. 4,166,093.This migration-inhibiting layer inhibits the migration of awater-soluble indicator by immobilizing it with a polymer mordant.However, the mordant often inhibits coloration of the indicator and,accordingly, it is not a fundamental solution to this problem. Besides,the preparation of this layer is difficult, thereby raising the cost ofthe analytical element.

It is also a problem that an aqueous liquid sample spotted on a porousspreading layer spreads too broadly in this spreading layer. In EP0,162,302A, hydrophilic cellulose derivatives and nonionic surfactantshaving an HLB value of more than 10 are disclosed as being effective asa spreading action controller. The hydrophilic cellulose derivative isdissolved in water and applied on the spreading layer. Since thesolubility of the nonionic surfactant in water is low, it is dissolvedin a mixture solvent of water and acetone.

On the other hand, colorimetry using an indicator is widely employed inthe field of clinical analysis. The indicator is usuallyo-Cresolphthalein Complexone of which the optimum pH for coloration isin the alkaline range, particularly higher than pH 10 where the bindingto calcium is stable. Also, it is known that the samples for clinicaldiagnosis, such as, blood, contain the calcium in a bound state, suchas, protein-bound calcium and acid-bound calcium, in addition to thecalcium in an ion state. All calcium in the sample is first ionized byan acid treatment, and thereafter, a color reaction is carried out(Clin. Chem. Vol. 29, p. 1497 (1983)). This method is, for example,described on pages 148 to 150 of "Jissen Rinsho Kagaku-Zoho Ban(Practical Clinical Chemistry-Revised and Enlarged Edition)" (Kitamuraet al., Ishiyaku Shuppan Kabushiki Kaisha, Japan 1982). An outline ofthis method is as follows. o-Cresolphthalein Complexone is dissolved ina small amount IN KOH, and glacial acetic acid is added to this solutionto prepare an acidic solution 1. An alkaline solution 2 containingpotassium acetate-HCl-diethylamine is separately prepared. The solution1 is added to a sample, and allowed to react. Subsequently, the solution2 is added to the reaction mixture, and color reaction proceeds.Recently, a one-step type reagent kit has also been available ("AgentCalcium", OINABBAT, USA). It has been reported that a linear relationbetween absorbance and concentration is obtained by allowing citricacid, its salt, or mixtures thereof, to coexist throughout the reaction(Japanese Patent Kokai No. 57-154058). However, in this patent, citricacid is used in a concentration of 0.01 to 0.05 M/l in an alkalinecondition, such as, pH 10 to 11, but this acid is not utilized foracidification of the sample nor the color reaction.

In the case of the analytical element for analysis of calcium, in orderto decompose the bound state of calcium, the pH of the upper layer ismade lower than 5, usually pH 1 to 2. On the other hand, the optimumreaction pH of preferable indicators, such as, o-Cresolphthalein isusually higher than pH 10. Since it is difficult for such a high pHlayer to coexist with the low pH layer in a thin analytical element, thecoloring reaction was set to proceed at about pH 5.5.Chlorophosphonazo-III and Arsenazo-III were used as the indicator.However, in this analytical element, the absorbance of the background ishigh, such as, higher than 2 and the measurement is carried out at along wave length (near 680 nm). Accordingly, the analytical accuracybecomes worse.

SUMMARY OF THE INVENTION

An object of the invention is to provide a method of preparing anintegral multilayer analytical element using a water-soluble indicatorwhere migration of the indicator does not occur and thereby the accuracyof measurement is maintained.

Another object of the invention is to provide a method of preparing anintegral multilayer analytical element using a water-soluble indicatoreasily without lowering the accuracy of the measurement.

Another object of the invention is to provide a method of preparing anintegral multilayer analytical element using a water-soluble indicatorinexpensively without lowering the accuracy of the measurement.

Another object of the invention is to provide a method of preparing anintegral multilayer analytical element capable of determinating totalcalcium concentration easily without pre-treatment by treating with acidin the analytical element and subsequently allowing to react to formcolor.

Another objection of the invention is to provide a method of preparingan integral multilayer analytical element using a water-solubleindicator capable of determinating total calcium concentration with highaccuracy.

Another object of the invention is to provide a method of easilypreparing an integral multilayer analytical element capable ofdeterminating total calcium concentration having the abovecharacteristics.

Another object of the invention is to provide an integral multilayeranalytical element capable of determinating total calcium concentrationhaving the above characteristics.

Still another object of the invention is to provide a method ofmeasuring total calcium concentration of an aqueous sample easily andrapidly by using such an integral multilayer analytical element.

The present inventors have investigated in order to achieve such objectsand found that the migration of a water-soluble indicator can beinhibited by dissolving a spreading action controller and/or an acid inan organic solvent which does not dissolve the water-soluble indicator,and incorporating the spreading action controller and/or the acid intothe porous spreading layer, such as, by the application of the abovespreading action controller and/or the acid solution on the spreadinglayer.

Thus, the present invention provides a method of preparing an integralmultilayer analytical element comprising a water-impermeablelight-transmissive support, a reagent layer containing a water-solubleindicator capable of reacting with an analyte to produce an opticallydetectable change, and a porous spreading layer containing a spreadingaction controller and/or an acid, superposed in this order, comprisingdissolving the spreading action controller and/or the acid in an organicsolvent which does not dissolve the above water-soluble indicator,incorporating this solution into the porous spreading layer, and dryingthis superposed material.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As the water-impermeable light-transmissive support, a known supportemployed in an usual multilayer analytical element may be employed. Sucha support is a film, a sheet or a flat plate having a thickness of about50 μm to about 1 mm, preferably, about 80 μm to about 0.3 mm and capableof transmitting the object light being the wave length range of about200 nm to about 900 nm. Such a support may be made from a polyester (forexample, polyethylene terephthalate or polycarbonate of bisphenol A), acellulose ester (for example, cellulose diacetate, cellulose triacetateor cellulose acetate propionate), or polystyrene. The optical propertyof the support may be controlled by suspending light-reflective orlight-absorptive particles, such as, titanium dioxide particles, bariumsulfate particles or carbon black therein. A known undercoating layer ora known adhesive layer may be provided on the surface of the support inorder to secure the adhesion of the support to the reagent layer, awater-absorption layer or the like, superposed on the support.

The reagent layer is water-absorptive and water-permeable and comprisesa hydrophilic polymer as a polymer binder and a reagent compositioncontaining at least an indicator capable of reacting with the analyte inan aqueous sample to produce an optically detectable change uniformlydispersed therein.

The hydrophilic polymer used in the reagent layer has a swelling ratioin the range from about 150% to about 2,000%, preferably, about 250% toabout 1,500% at a water absorption at 30° C. Examples of the hydrophilicpolymer are gelatins including acid treated gelatin and deionizedgelatin, gelatin derivatives, such as, phthalated gelatin andhydroxyalkyl acrylate grafted gelatin, agarose, pullulan, pullulanderivatives, polyacrylamide, polyvinyl alcohol and polyvinylpyrrolidone. They are disclosed in EP 0,119,861A and EP 0,142,849A.Preferable hydrophilic polymers are usually gelatins, gelatinderivatives, polyacrylamide and polyvinyl alcohol, deionized gelatinbeing is the most preferable.

The thickness of the reagent layer in the dry state is about 5 μm toabout 50 μm, preferably about 7 μm to about 30 μm. The coating weight ofthe reagent layer itself is about 5 g/m² to about 50 g/m², preferablyabout 7 g/m² to about 30 g/m².

The indicator reacts with an analyte to produce an optically detectablechange. The wave length used for detection is not limited to the visibleregion, and includes the ultraviolet region and infrared region. Thisindicator is water-soluble, and the method of the invention isparticularly effective when solubility of the indicator is higher than 5mg/100 g water at 25° C. On the other hand, it is also necessary that itnot dissolve in the organic solvent used for the spreading actioncontroller. Such an indicator is selected from known ones and it mayform a complex with an analyte.

The analyte includes calcium, magnesium, inorganic phosphorus and iron.These exist in an ionic state, a salt of a fatty acid, a proteinconjugate or the like. The above inorganic phosphorus exists in thestate of a phosphate acid ion or a phosphate salt. The liquid samplecontaining such a sample includes various biological fluids, foods,drinks, liquors and medicines.

The indicators for the analysis of calcium are, for example, describedin "Dotite Reagents Catalog" (Dojindo Laboratories, Kumamoto, Japan,1980). Examples of the indicator are o-Cresolphthalein Complexone(3,3'-bis[[di(carboxymethyl)-amino]methyl]-o-cresolphthalein[2411-89-4], optimum pH; about 10.5), Eriochrome Black T(monosodium saltof 1-(1-hydroxy-2-naphthylazo)-6-nitro-2-hydroxynaphthalene-4-sulfonicacid [1787-61-7]), Methylthymol Blue Complexone (tetrasodium salt of3,3'-bis[[di(carboxymethyl)amino]methyl]thymolphthalein [1945-77-3]),Thymolphthalein Complexone(3,3'-bis[[di(carboxymethyl)amino]-methyl]thymolphthalein [1913-93-5]),Arsenazo-III(2,7-bis[(2-arsonophenyl)azo]-1,8-dihydroxynaphthalene-3,6-disulfonicacid [1668-00-4]) andChlorophosphonazo-III)2,7-bis[(4-chloro-2-phosphonophenyl)azo]-1,8-dihydroxynaphthalene-3,6-disulfonicacid [1914-99-4]). The figures in the brackets represent chemicalAbstracts Registry Numbers. Among these, o-Cresolphthalein Complexoneand Arsenazo-III are preferred for the most accurate determination ofcalcium.

In the multilayer analytical element for analysis of calcium, a maskingagent for masking magnesium ion is preferably incorporated. Such amasking agent includes 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonicacid, 8-hydroxyquinoline sulfate and the like. Suitable layers forincorporating the masking agent are the reagent layer and/or its upper(far from the support) adhesive layer or the spreading layer. Thecontent of the masking agent is about 1.5 to about 10 times, preferablyabout 2 to about 5 times, the content of the indicator.

The indicator for analysis of magnesium includes Xylidyl BlueIR(monosodium salt of3-[[3-(2,4-dimethylphenyl-carbamoyl)-2-hydroxy-l-naphthyl]azo]-4-hydroxybenzenesulfonicacid [14936-97-1]), and the indicator for analysis of iron includesBathophenanthroline disulfonic acid.2Na salt(disodium salt of4,7-bis(4-sulfophenyl)-1,10-phenanthroline). The indicator for analysisof inorganic phosphorus includes p-(methylamino)phenol sulfate.

The reagent composition may be incorporated into two or more layers, forexample, the reagent layer and a water-absorption layer.

In the reagent layer, a pH buffer may also be incorporated in order tomaintain the reaction with the indicator at its optimum. This pH isdifferent according to each reaction, and in the case of analyzingcalcium using o-Cresolphthalein as the indicator, the optimum pH is at 9to 10.5. The pH buffers suitable for integral multilayer analyticalelements are described in "Kagaku Benran Kiso-Hen" pp 1312-1320,Maruzen, Tokyo, 1966, R. M. C. Dawson et al, "Data for BiochemicalResearch", 2nd Ed., pp 476-508, Oxford at the Clarendon Press, 1969,"Biochemistry", Vol. 5, from p. 467, 1966 and "Analytical Biochemistry",Vol. 104, pp 300-310, 1980. The pH buffer in the range of pH 8.0 to11.0, particularly, pH 9.0 to 10.5, includes buffers containingtris(hydroxymethyl)aminomethane, buffers containing a phosphate, bufferscontaining a borate, buffers containing a carbonate, buffers containingglycine and the like. Examples are N,N-bis(2-hydroxyethyl)glycine(Bicine), sodium or potassium salt ofN-2-hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid (HEPPS),sodium or potassium salt of N-2-hydroxy-ethylpiperazine-N'-3-sulfonicacid (EPPS), sodium or potassium salt of3-(cyclohexylamino)-l-propanesulfonic acid (CAPS), sodium or potassiumsalt of N-[tris(hydroxymethyl)methyl]-3-amino-propanesulfonic acid(TAPS), sodium or potassium salt ofN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and acombination of any of them and an acid, an alkali or a salt. Preferablebuffers include Tris-sodium borate, Bicine, HEPPS, sodium salt of HEPPS,EPPS, sodium salt of EPPS, CAPS, sodium salt of CAPS, TAPS and sodiumsalt of TAPS. When gelatin or its derivative is used as the hydrophilicpolymer, the pH buffer containing boric acid or sodium borate ispreferable in that this layer can suitably be hardened by cross-linkingusing a crosslinking agent having vinyl sulfone structure. This gelatinor its derivative layer are stable when applied and quantitativeanalysis can be performed with high accuracy. A known base polymer isalso usable as a pH buffer. A surfactant, such as, the nonionicsurfactant described later, may also be added to the reagent layer.

The reagent layer is preferably transparent, but its optical propertymay be controlled by suspending a small amount of titanium dioxideparticles, barium sulfate particles or carbon black therein.

The porous spreading layer includes spreading layers of woven fabricdisclosed in U.S. Pat. No. 4,292,272 and GB No. 2,087,074A, such as,plain weaves including broad cloth and poplin, spreading layers ofknitted fabric disclosed in EP 0,162,302A, such as, tricot fabric,double tricot fabric and milanese fabric, spreading layers composed of apaper containing fibrous pulp of an organic polymer disclosed inJapanese Patent Kokai No. 57-148250, membrane filters (blushed polymerlayer) as disclosed in U.S. Pat. No. 3,992,158, continuousmicrospaces-containing porous layers where polymer particulates, glassparticulates or diatomaceous earth are dispersed in a hydrophilicpolymer binder, and continuous microspaces-containing porous layerswhere polymer particulates, glass particulates, etc. are joined so as tocontact with each other at a point by using a polymer adhesive whichdoes not swell in water (three-dimensional lattice structure layer)disclosed in U.S. Pat. No. 4,258,001.

In order to raise the adhesive force, the above fibrous porous spreadinglayer, such as, woven fabrics, knitted fabrics and papers may be madehydrophilic by a physical activation treatment, such as, glow dischargeor corona discharge disclosed in GB 2,087,074A, a chemical treatment,such as, washing, degreasing and immersing in a hydrophilic polymersolution, disclosed in U.S. Pat. No. 4,292,272 and GB 2,087,074A, or acombination thereof.

The spreading action controller is incorporated in the porous spreadinglayer. The spreading action controller controls the spreading of anaqueous liquid sample in the spreading layer so as not to spread toobroadly, and is selected from hydrophilic polymers or nonionicsurfactants.

The hydrophilic polymers are polyvinyl pyrrolidone, polyvinyl alcohol,polyacrylamide, polyacrylic acid, hydrophilic cellulose derivatives, andthe like. The hydrophilic cellulose derivatives are cellulose ethers inwhich a part of or whole hydroxyl groups of cellulose are converted toethers by introducing lower alkyl groups having a carbon number of 1 to3 or hydroxyl group-substituted lower alkyl groups having a carbonnumber of 1 to 4. Examples of such a cellulose ether are watersoluble,and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose and hydroxybutyl methylcellulose.Preferable hydrophilic polymers are polyvinyl pyrrolidone, polyvinylalcohol and water-soluble cellulose ethers. Two or more hydrophilicpolymers may be combined in use. The content of the hydrophilic polymerin the porous spreading layer is about 0.5 to about 15 g/m², preferablyabout 0.7 to about 10 g/m².

The nonionic surfactants are polyhydric alcohol ester ethylene oxideadducts (condensate), polyethylene glycol monoesters, polyethyleneglycol diesters, higher alcohol ethylene oxide adducts (condensate),alkylphenol ethylene oxide adducts (condensate) higher fatty acidalkanol amides and the like. Examples of the nonionic surfactant are:

POE (20) sorbitan monooleate

POE (10) sorbitan monooleate

POE (4) sorbitan tristearate

POE (4) trioleate

POE (30) stearate

POE (40) stearate

POE (100) stearate

PEG (400) monostearate

PEG (400) monolaurate

PEG (1000) dilaurate

PEG (1540) distearate

Lauryl alcohol EO 6 moles condensate

Lauryl alcohol EO 10 moles condensate

Lauryl alcohol EO 30 moles condensate

Oleyl alcohol EO 20 moles condensate

Cetyl alcohol EO 20 moles condensate

POE (10) octylphenyl ether

POE (15) octylphenyl ether

POE (30) octylphenyl ether

POE (12) nonylphenyl ether

POE (20) nonylphenyl ether

Triethanolamine oleate

As used herein POE=polyethylene oxide, PEG=polyethylene glycol,EO=ethylene oxide. The number in parentheses represents the condensationnumber of ethylene oxide units.

Two or more nonionic surfactants may be combined in use. When a nonionicsurfactant is combined with a hydrophilic polymer, HLB value of thenonionic surfactant is preferably more than 10. The content of nonionicsurfactant in the porous spreading layer is about 0.1 to about 3 g/m₂,preferably about 0.2 to 2 g/m².

In the cases of analytical elements for analysis of calcium, an acid fordecomposing the calcium compounds in a sample is preferably dissolved inan organic solvent which does not dissolve the indicator selected fromthose previously mentioned. The calcium compounds include protein-bound,such as, albumin-bound calcium compounds and acid-bound calciumcompounds, such as calcium phosphate. Heretofore, the error caused bycalcium phosphate has not been recognized as a problem. The presentinventors have found that the analytical value of calcium concentrationbecomes lower with increasing phosphate concentration. In the case ofthe analytical element of the invention, the exact analytical value canbe obtained, though phosphate concentration is relatively high.

In order to decompose protein-bound calcium, the pH is preferably lowerthan 4, while, in order to decompose calcium phosphate, the pH ispreferably at about 1 to 2. Since a pH buffer is not incorporated in theporous spreading layer, the pH of this layer can be adjusted to such alow range by adding a small amount of an acid. The acid may beincorporated into the porous spreading layer in the form of asuspension. Examples of the acid are fatty acids, such as,monocarboxylic acids, including acetic acid and dicarboxylic acids,including malonic acid, glutaric acid, adipic acid and pimelic acid,halogen-substituted fatty acids, such as, monochloroacetic acid andtrichloroacetic acid, aromatic carboxylic acids, such as, phthalic acid,aliphatic hydroxycarboxylic acids, such as, lactic acid, malic acid andcitric acid, aromatic hydroxycarboxylic acids, such as, salicylic acidand sulfonic acids, such as, sulfosalicylic acid and1,5-naphthalenedisulfonic acid. Two or more acids may be combined, ifnecessary. In the spreading layer of the analytical element for analysisof calcium, a quaternary amine polymer or the like may be incorporated,and inorganic phosphorus, such as, phosphate ions released by the acidtreatment are trapped by this polymer.

In such an analytical element, the bound state calcium, such as,protein-bound calcium and calcium phosphate compounds are decomposed byaction of the acid incorporated in the spreading layer to generatecalcium ion. While the calcium in an ion state passes through thespreading layer as it is. The acid accompanied with calcium ion isneutralized with the pH buffer incorporated in the reagent layer orother layers, and adjusted around the optimum pH of color reaction.Then, total calcium ion reacts with the indicator to produce adetectable optical change and the object calcium content can bedetermined by detecting this optical change. Other layers may beincorporated in the analytical element.

For example, a water-absorption layer may be provided between thesupport and the reagent layer. The water-absorption layer is mainlycomposed of a hydrophilic polymer which absorbs water to swell, and itabsorbs the water of an aqueous liquid sample which reaches the surfaceof this layer. In the case of a Whole blood sample, it accelerates thepermeation of the blood plasma component into the reagent layer. Thehydrophilic polymer is selected from those mentioned previously. Ingeneral, gelatin, a gelatin derivative, polyacrylamide and polyvinylalcohol are preferable, gelatin being the most preferred. The drythickness of the water-absorption layer is about 3 μm to about 100 μm,preferably about 5 μm to about 30 μm. The coating weight of thewater-absorption layer itself is about 3 g/m² to about 100 g/m²,preferably about 5 g/m² to about 30 g/m². A pH buffer selected fromthose described previously or a base polymer may be incorporated in thewater-absorption layer. A nonionic surfactant and a known mordant orpolymer mordant may also be incorporated.

A hydrophilic nonporous intermediate layer may be provided between theporous spreading layer and the reagent layer. This layer inhibitspermeation of proteins, such as, albumin and globulin, and it iscomposed of a hydrophilic polymer as mentioned previously or crosslinkedthereof. Preferable hydrophilic polymers are gelatin, gelatinderivatives, polyacrylamide and polyvinyl alcohol, and gelatin,particularly deionized gelatin is the most preferable. Examples ofcrosslinking agents are vinylsulfonyl crosslinking agents, such as,1,2-bis(vinylsulfonylacetamide)ethane and bis(vinylsulfonylmethyl) etherand aldehydes for gelatin, and aldehydes and two glycidyl groupscontaining epoxy compounds for methallyl alcohol copolymer.

The dry thickness of the intermediate layer is about 3 μm to about 20μm, preferably about 5 μm to 15 μm. In this layer, a pH buffer mentionedpreviously or a known base polymer and a nonionic surfactant may beadded. Light-reflecting particles, such as, titanium dioxide particlesor barium sulfate particles may be suspended, and it also functions as alight-reflecting layer. The light-reflecting layer may be providedseparately. The dry thickness of the light-reflecting layer is about 5μm to about 50 μm, preferably about 7 μm to about 30 μm. The coatingweight of the light-reflecting layer itself is about 5 g/m² to about 50g/m², preferably about 7 g/m² to about 30 g/m².

An adhesive layer may be provided in order to fortify the adhesive forceto the spreading layer. This layer is composed of the hydrophilicpolymer mentioned previously, such as gelatin, and its dry thickness isabout 0.5 μm to about 5 μm.

The method of the invention is characterized by incorporating thespreading action controller or the acid mentioned previously using anorganic solvent which does not dissolve the water-soluble indicatorincorporated in the reagent layer. Such a solvent is usually a polarsolvent having a boiling point of lower than 100° C, and includesaliphatic alcohols, such as, methanol, ethanol, propanol, butanol andisopropanol, dialkylketones, such as, acetone and methyl ethyl ketone,dialkyl ethers, such as, diethyl ether, aliphatic cyclic ethers, suchas, tetrahydrofuran and dioxane. Some nonpolar solvents, such as,acetonitrile, benzene and hexane are also included. Among these,aliphatic alcohols are preferable, and low toxic alcohols, such as,ethanol, propanol, butanol and isopropanol are particularly preferablein view of working circumstances. For example, when o-Cresolphthalein isemployed as the indicator for analysis of calcium, the above alcoholsare suitable. This compound itself is slightly soluble in water butreadily soluble in alcohols. However, near its optimum pH (pH 10.5), itsis readily soluble in water but insoluble in alcohols.

The concentration of the spreading action controller is preferably high,such as, about 0.2% to about 10%, particularly, about 0.3% to about 7%.A small amount of water may be added to the spreading action controllersolution so as to prevent migration of the water-soluble indicator tothe spreading layer. The spreading action controller solution may beapplied or sprayed uniformly on the spreading layer. When both ahydrophilic polymer and a nonionic surfactant are incorporated, a mixedsolution of the two may be used. They may also be incorporatedseparately. In the case of the acid, a suspension of the acid ma be usedinstead of its solution.

The application or spraying is preferably carried out after thespreading layer is incorporated into the analytical element. When thespreading layer is a membrane filter (blushed polymer layer), knittedfabric or woven fabric, the solution or suspension may be added to thespreading layer prior to its lamination. However, in this case, it isdifficult to adjust the content of the spreading action controller orthe acid to a prescribed value.

After the application or spraying, the analytical element is dried underreduced pressure, by air-drying or the like.

Other processes for preparing the analytical element of the inventionmay be used.

The integral multilayer analytical element of the invention ispreferably cut into square or circular pieces having a side or diameterof about 15 mm to about 30 mm, and put in a slide frame disclosed inU.S. Pat. No. 4,169,751, Japanese Patent No. 57-63452, U.S. Pat. No.4,387,990 and Japanese Utility Model Kokai No. 58-32350, PCT applicationWO 83/00391, etc. for use.

The measurement is carried out, for example, according to the mannerdisclosed in the specifications of the above-mentioned patents. About 5μl to about 30 μl, preferably about 8 μl to about 15 μl of an aqueoussample is spotted on the spreading layer, and incubated at a definitetemperature in the range of about 20° C to about 45° C for 1 to 10minutes. Thereafter, a detectable change, such as, the color change orcoloring in the multilayer analytical element is measured from the sideof the support through reflection photometry, and the subject componentin the sample is determined by the principle of colorimetry. When thismeasurement is carried out by using the chemical-analytical apparatusdisclosed in U.S. Pat. No. 4,488,810 and U.S. Pat. No. 4,424,191, highlyaccurate results can easily be obtained by a simple operation.

In the method of the present invention, the migration of thewater-soluble indicator in the reagent layer at the time ofincorporating the spreading action controller or the acid into thespreading layer is inhibited by using a particular organic solvent.Migrations of the indicator and colored material to the spreading layerare also lowered by the spreading action controller, and, as a result,the analytical accuracy of the analytical element is improved. In thecase of the analytical element for analysis of calcium, permeation ofthe acid into other layers than the spreading layer is inhibited, andpermeation of a pH buffer into other layer(s) into the spreading layeris also inhibited by using a particular organic solvent. As a result,effective decomposition of calcium compounds in a sample and effectivecoloration can be achieved.

EXAMPLES Example 1

The support employed was a colorless transparent polyethyleneterephthalate (PET) film having a thickness of 180 μm. The followinglayers were successively coated and dried to form a laminate.

    ______________________________________                                        Water-absorption layer:                                                       Deionized gelatin         4.8     g/m.sup.2                                   nonylphenoxypolyethoxyethanol                                                                           0.11    g/m.sup.2                                   (containing 10 hydroxyethylene units on average)                              1,2-bis(vinylsulfonylacetamide)ethane                                                                   0.5     g/m.sup.2                                   The aqueous solution was adjusted to                                          pH 6.5 by using NaOH.                                                         Reagent layer:                                                                Deionized gelatin         23.9    g/m.sup.2                                   polyoxyethylene nonyl phenyl ether                                                                      0.41    g/m.sup.2                                   (containing 10 hydroxyethylene units on average)                              CAPS                      3.81    g/m.sup.2                                   o-Cresolphthalein Complexone                                                                            0.15    g/m.sup.2                                   8-hydroxyquinolin-5-sulfonic acid                                                                       0.54    g/m.sup.2                                   The aqueous solution was adjusted to                                          pH 10.6 by using NaOH.                                                        Adhesive layer:                                                               Deionized gelatin         1.46    g/m.sup.2                                   Polyoxyethylene nonyl phenyl ether                                                                      0.10    g/m.sup.2                                   (containing 10 hydroxyethylene units on average)                              titanium dioxide particles                                                                              0.85    g/m.sup.2                                   ______________________________________                                    

The adhesive layer was uniformly dampened with water, and a PET tricotfabric cloth knitted from 100S PET spun yarn having a mean thickness of250 μm was pressed to laminate thereon as the spreading layer.Subsequently, the following polymer ethanol solution was applied on thespreading layer and dried to obtain the integral multilayer analyticalelement for analysis of calcium.

    ______________________________________                                        Polymer ethanol solution:                                                     ______________________________________                                        Polyvinyl pyrrolidone     1.01 g/m.sup.2                                      (mean molecular weight 360,000)                                               polyoxyethylene nonyl phenyl ether                                                                      2.11 g/m.sup.2                                      (containing 40 hydroxyethylene units on average)                              dissolved in ethanol.                                                         ______________________________________                                    

Comparative Example 1

An integral multilayer analytical element for analysis of calcium wasprepared in the same manner as Example 1, except for the following: Thecontent of o-Cresolphthalein Complexone in the reagent layer wasincreased from 0.15 g/m² to 0.46 g/m², and the content of8-hydroxyquinoline-5-sulfonic acid in the reagent layer was increasedfrom 0.54 g/m² to 1.65 g/m². Instead of the polymer ethanol solution,the following aqueous polymer solution was employed.

    ______________________________________                                        Aqueous polymer solution:                                                     ______________________________________                                        Methyl cellulose           5.75   g/m.sup.2                                   (viscosity of 2% aqueous solution at 20° C.; 100 cps)                  nonylphenoxypolyethoxyethanol                                                                            7.44   g/m.sup.2                                   (containing 40 hydroxyethylene units on average)                              titanium dioxide particles 15.5   g/m.sup.2                                   dissolved and suspended in water, and applied.                                ______________________________________                                    

Example 2

The integral multilayer analytical elements prepared in Example 1 andComparative Example 1 were evaluated by using a commercial control serum"Monitrol IX" (DADE, U.S.A.), and the results are shown in Table 1. Asshown in the Table, in the case of the analytical element of Example 1,dispersion of the measured values are remarkably small.

                  TABLE 1                                                         ______________________________________                                        Example 1              Comparative 1                                                   Measured               Measured                                      Run No.  Value mg/dl   Run No.  Value mg/dl                                   ______________________________________                                         1       7.7            1       7.9                                            2       7.7            2       7.6                                            3       7.7            3       7.9                                            4       7.8            4       7.7                                            5       7.9            5       7.7                                            7       7.7            7       7.6                                            8       7.8            8       7.6                                            9       7.8            9       7.4                                           10       7.6           10       7.6                                           11       7.8           11       7.6                                           12       7.7           12       7.9                                           13       7.7           13       7.6                                           14       7.7           14       7.7                                           15       7.8           15       7.7                                           16       7.8           16       7.7                                           17       7.8           17       7.4                                           18       7.7           18       7.6                                           19       7.7           19       7.4                                           20       7.6           20       7.5                                           21       7.6           21       7.5                                           22       7.7           22       7.7                                           23       7.8           23       7.6                                           24       7.8           24       7.5                                           25       7.7           25       7.7                                           Average  7.74          Average  7.63                                          SD       0.0757        SD       0.1406                                        CV       0.98%         CV       1.8%                                          ______________________________________                                    

Example 3

The following layers were successively applied on a colorlesstransparent PET film having a thickness of 180 μm, and dried to form alaminate.

    ______________________________________                                        Reagent layer:                                                                Deionized gelatin         16.8    g/m.sup.2                                   nonylphenoxypolyethoxyethanol                                                                           1.1     g/m.sup.2                                   (containing 10 hydroxyethylene units on average)                              CAPS                      2.8     g/m.sup.2                                   o-Cresolphthalein Complexone                                                                            0.15    g/m.sup.2                                   8-hydroxyquinoline-5-sulfonic acid                                                                      0.56    g/m.sup.2                                   The aqueous solution was adjusted to pH 10.6 by                               using NaOH.                                                                   Adhesive layer:                                                               Deionized gelatin         1.46    g/m.sup.2                                   nonylphenoxypolyethoxyethanol                                                                           0.10    g/m.sup.2                                   (containing 10 hydroxyethylene units on average)                              titanium dioxide          0.85    g/m.sup.2                                   The aqueous solution was adjusted to pH 10.6                                  by using NaOH.                                                                ______________________________________                                    

The adhesive layer was uniformly dampened with water, and a tricotknitted fabric cloth was pressed to laminate thereon as the spreadinglayer. Subsequently, the following polymer ethanol solution was appliedon the spreading layer, and dried to obtain the integral multilayeranalytical element for analysis of calcium.

    ______________________________________                                        Polymer ethanol solution:                                                     ______________________________________                                        Polyvinyl pyrrolidone     4.1 g/m.sup.2                                       (mean molecular weight 360,000)                                               nonylphenoxypolyethoxyethanol                                                                           8.6 g/m.sup.2                                       (containing 10 hydroxyethylene units on average)                              trichloroacetic acid      0.9 g/m.sup.2                                       dissolved in ethanol.                                                         ______________________________________                                    

Comparative Example 2

An integral multilayer analytical element was prepared in the samemanner as Example 3 except trichloroacetic acid was not added to thepolymer ethanol solution.

Example 4

Calcium concentrations of the following Solution 1 and Solution 2 weremeasured by using the analytical elements prepared in Example 3 andComparative Example 2.

Solution 1: Aqueous solution containing 10 mg/ml of calcium (CaCl₂ wasused).

Solution 2: Aqueous solution containing 10 mg/ml of calcium (CaCl₂ wasused) and 10 mg/l of phosphorus (Na₂ HPO₄ was used). Since the solutionbecame turbid, it was stirred vigorously before each sampling.

The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                    Example 3                                                                             Comparative 2                                             ______________________________________                                        Solution 1    10.0 mg/dl                                                                              10.0 mg/dl                                            Solution 2     9.4 mg/dl                                                                               6.2 mg/dl                                            ______________________________________                                    

As shown in the table, the negative error in the case of using Example 3in the presence of phosphorus is less than one sixth of that ofComparative product 2.

Example 5

An integral multilayer analytical element was prepared in the samemanner as Example 3, except 0.9 g/m² of trichloroacetic acid in thepolymer ethanol solution was replaced by 0.6 g/m² of citric acid. Thecalcium concentrations of Solution 1 and Solution 2 were measured byusing this analytical element, and similar results to Example 4 wereobtained.

What is claimed is:
 1. A method of preparing an integral multilayeranalytical element for analysis of calcium compounds comprising, in thisorder, a water-impermeable light-transmissive support, a reagent layercontaining a water-soluble indicator capable of reacting with calcium toproduce an optically detectable change, and a porous spreading layercontaining an acid capable of decomposing the calcium compounds in asample which comprises dissolving said acid in an organic solvent whichdoes not dissolve said water-soluble indicator, incorporating thissolution into said porous spreading layer, and drying the layer, whereinsaid integral multilayer analytical element contains a pH buffer capableof maintaining the pH of the reaction with said indicator at its optimumpH incorporated in said reagent layer or an intermediate layer betweensaid reagent layer and said porous spreading layer.
 2. The method ofclaim 1 wherein said compounds are protein-bound calcium and calciumphosphate.
 3. The method of claim 2 wherein said acid is selected fromthe group consisting of monocarboxylic acids, dicarboxylic acids,halogen-substituted fatty acids, aromatic carboxylic acids, aliphatichydroxycarboxylic acids, aromatic hydroxycarboxylic acids, and sulfonicacids.
 4. The method of claim 2 wherein said acid is selected from thegroup consisting of acetic acid, malonic acid, glutaric acid, adipicacid, pimelic acid, monochloroacetic acid, trichloroacetic acid,phthalic acid, lactic acid, malic acid, citric acid, salicylic acid,sulfosalicylic acid and 1,5-naphthalenedisulfonic acid.
 5. The method ofclaim 1 wherein said solution contains a spreading action controller. 6.The method of claim 5 wherein said spreading action controller is one orboth of a hydrophilic polymer and a nonionic surfactant.
 7. The methodof claim 6 wherein said hydrophilic polymer is selected from the groupconsisting of polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylicacid, methyl cellulose and ethyl cellulose, and said nonionic surfactantis selected from the group consisting of polyhydric alcohol esterethylene oxide adducts, polyethylene glycol monoesters, polyethyleneglycol diesters, higher alcohol ethylene oxide adducts, alkylphenolethylene oxide adducts and higher fatty acid alkanol amides.
 8. Themethod of claim 1 wherein said organic solvent is a polar organicsolvent having a boiling point of lower than 100° C.
 9. The method ofclaim 8 wherein said polar organic solvent is selected from the groupconsisting of aliphatic alcohols, dialkylketones, dialkyl ethers andaliphatic cyclic ethers.
 10. The method of claim 9 wherein said polarorganic solvent is selected from the group consisting of methanol,ethanol, propanol, butanol and isopropanol.
 11. The method of 1 whereinsaid indicator is o-Cresolphthalein Complexone, Arsenazo-III orChlorophosphonazo-III.